Dale T. Lindgren and Dermot P. Coyne
Differences in potato leafhopper (Empoasca fubae Harris) injury symptoms were noted in 22 cultivars or lines of dry beans (Phaseolus vulgaris L.) in a 1991 field trial at North Platte, Neb. Seed yield, biomass, and plant injury symptoms were recorded. The same 22 dry bean cultivars or lines were planted in a split-plot design, with main plots protected (sprayed with insecticide) vs. unprotected (not sprayed) and cultivars or lines as subplots in 1992 and 1993. Significant differences were observed between cultivars/lines for leafhopper injury and yield in all 3 years. `Tacaragua' (black-seeded) and pinto `Sierra' were highly resistant to leafhoppers, with no visual leafhopper injury symptoms in all 3 years. Significant negative correlation coefficients between leafhopper injury symptoms and yield were recorded in the protected (4.50) and unprotected (-0.33) plots in 1993 but only in the unprotected (-0.46) plots in 1992. A cultivar x spray interaction response to leafhoppers occurred in 1992 but not in 1993. The degree of leafhopper injury symptoms varied between years.
Dermot P. Coyne, Lisa Sutton, and Debra Fujimoto
No sources of resistance to Xanthomonas campestris pv. cucurbitae (X.c.c.) in the Cucurbita species have been reported. Cultivars, breeding lines, landraces, and PI lines of 5 Cucurbita species were screened for resistance to X.c.c. in 3 greenhouse tests (GH). A `florist`s frog' was used to inoculate the first fully expanded leaves using a X.c.c. suspension (107 CFU/ml). The disease reaction was recorded as the percentage of inoculated leaf area with necrotic lesions and/or chlorosis. Butternut (b n) breeding lines were also evaluated for reaction to X.c.c. under uniform natural in feetion in 2 field (F) trials. A randomized complete block design was used in all GH and F experiments. C. moschata Nebr. BN PM1-88-8 and C. martinezii had high resistance to X.c.c. All other entries in all tests were susceptible. BN PM-88-8 is an early maturing small stable (no crookneck fruit) near-round BN type squash. The fruit are resistant to black–rot, but the leaves are susceptible to powdery mildew. BN PM-88-8 is ideal for microwave cooking because of its near-round shape permitting more uniform cooking. Release is expected in 1990.
Mohamed F. Mohamed and Dermot P. Coyne
Common bacterial blight, incited by Xanthomonas campestris pv. phaseoli (Smith) Dye (Xcp), is a serious disease of common beans (Phaseolus vulgaris L.). Three experiments were conducted twice in growth chambers at 26 ± 1C under short (10 hours light/14 hours dark) and long (16 hours light/8 hours dark) photoperiods to determine the influence of these photoperiods, flower bud removal, pod development, and pre- and post-inoculation photoperiods on the reaction of common beans to Xcp. In one test, `PC-50' (susceptible; S) flowered earlier and was more susceptible to Xcp under the short photoperiod than under the long photoperiod. BAC-6 (resistant; R) flowered at the same time under both photoperiods but developed rapid leaf chlorosis (RLC) (hypersensitive reaction) under long photoperiods. Flowering and disease reactions to Xcp by XAN-159 (R) were similar under both photoperiods. In a second test, daily removal of flower buds of `PC-50' decreased its susceptibility to Xcp under the short photoperiod. RLC of inoculated leaves of BAC-6 occurred during flowering and pod development under both photoperiods. XAN-159 expressed a high level of resistance to Xcp but showed RLC at later pod development stages. In a third test, the disease reaction of `PC-50' was affected by the particular photoperiod applied post-inoculation but was not influenced by the photoperiod applied before inoculation with Xcp. The implications of these results in breeding beans for resistance to Xcp are discussed.
Zhanyuan Zhang, Dermot P. Coyne, and Amitava Mitra
Factors influencing Agrobacterium tumefaciens-mediated transformation of common beans (Phaseolus vulgaris L.) were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system to develop a repeatable transformation protocol. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains—A2760 and EHA105—were used, with emphasis on the former due to its overall higher infection rate. Eleven common-bean genotypes were compared for susceptibility to strain A2760 or EHA105. The pinto bean `Othello' was used extensively in testing different transformation conditions. Factors significantly affecting transformation rate were Agrobacterium × host interactions, explant maturity, preculture and cocultivation conditions, and selection schemes, based on transient GUS gene expression. The best transformation conditions were the use of susceptible genotypes and explants derived from mature seeds, preconditioning of explants in a medium containing 20 μmol of benzyladenine (BA) in darkness or on a filter paper, dipping explants in high concentrations of Agrobacterium cell suspension (OD650 = 0.8-1.0) followed by a long-term (6-day) cocultivation period on a semisolid agar medium in the presence of cytokinin or 3-day cocultivation on a moistened filter paper, and the use of lethal levels of selective agents. About 4% of explants, or 14% of regenerated shoots or buds, were putatively transgenic, as indicated by GUS blue staining throughout the entire shoot or bud, after explants were transformed with Agrobacterium strain A2760 using an optimized protocol.
James S. Beaver, James R. Steadman, and Dermot P. Coyne
Field reaction of 25 red mottled bean (Phaseolus vulgaris L.) genotypes to common bacterial blight [Xanthomonas campestris pv. phaseoli (Smith) Dye] was evaluated in Puerto Rico over 2 years. The average disease severity (percent leaf area with symptoms) was similar over years. The determinate red mottled genotypes had almost twice as much disease as indeterminate genotypes. Eight of the indeterminate genotypes had significantly less disease than the mean of the field experiments. These genotypes may serve as useful sources of resistance to common bacterial blight. The size of the chlorotic zone around necrotic lesions varied between growing seasons, showing that environment can influence the expression of common bacterial blight symptoms.
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
Few studies on embryogenesis in common bean (Phaseolus vulgaris L.) have been reported and only the early stages of somatic embryogenesis were observed. Dry seeds from two common bean lines were germinated in darkness on L-6 medium containing 4% sucrose, 0.2 g casein hydrolysate /liter and 2.0 g phytagel /liter. The medium for seed germination was supplemented with 0, 2, 4 or 6μM forchlorfenuron (CPPU). Explants from cotyledonary leaves, petioles, hypocotyls and shoot apices were prepared from 14 day-old seedlings. Callus was derived from explant cultures incubated in darkness at 26C on the medium containing 4 μM 2,4-D and 1 μM Kinetin. The callus was transferred after 4 weeks into 125 ml Erlenmeyer flasks containing 50 ml liquid medium and placed on a gyrotary shaker (120 rpm) under cool-white light (12 μmol.m-2.s-1). The liquid medium was used with 2, 4 or 6 μM of 2,4-D alone or with zeatin supplements at relative concentrations of 0.25 and 0.5. Up to 200 somatic embryos from 40 to 50 mg callus inoculations were induced after 4 to 5 weeks. Callus derived from seedlings grown on CPPU-containing medium gave more repetitive somatic embryos. Cotyledonary stage embryos with clear bipolar structure were observed only from callus derived from seedlings grown on CPPU when transferred to suspension cultures containing 2,4-D and zeatin. All somatic embryos differentiated strong roots and some developed leaf-like structures on conversion medium.
Soon O. Park, Dermot P. Coyne, and James R. Steadman
Bean rust, caused by Uromyces appendiculatus, is a major disease of common bean (Phaseolus vulgaris). The objective was to identify RAPD markers linked to the gene (Ur-7) for specific resistance to rust race 59 using bulked segregant analysis in an F2 segregating population from the common bean cross GN1140 (resistant to rust) × Nebraska #1 (susceptible to rust). A single dominant gene controlling specific resistance to race 59 was found in the F2 and was confirmed in the F3. Seven RAPD markers were detected in a coupling-phase linkage with the Ur-7 gene. Coupling-phase RAPD markers OAA11.500, OAD12.550, and OAF17.900 with no recombination to the Ur-7 gene were found. Three RAPD markers were identified in a repulsion-phase linkage with the Ur-7 gene among the three markers at a distance of 8.2 cM. This is the first report on RAPD markers linked to the Ur-7 gene in common bean. The RAPD markers linked to the gene for specific rust resistance of Middle American origin detected here, along with other independent rust resistance genes from other germplasm, could be used to pyramid multiple genes into a bean cultivar for more-durable rust resistance.
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
Dry seeds from two lines of common bean (Phaseolus vulgaris L.) and one cultivar of faba bean (Vicia faba L.) were germinated on Murashige and Skoog (MS) medium containing B vitamins, 30 g sucrose/liter, and either 2.5, 5.0, or 7.5 μm benzyladenine (BA). Axenic seed cultures were grown at 22 to 24C in darkness and under continuous light from cool-white fluorescent tubes (40 μmol·m-2·s-1). Explant tissues were prepared from cotyledonary nodes (CN) and primary nodes (PN) of 14-day-old seedlings. Explants were cultured on corresponding seedling growth medium and maintained under continuous cool-white light (40 μmol·m-2·s-1). The percentages of CN and PN (in one line of common bean) explants that regenerated shoots and the number of shoots per explant (in all germplasm) were highest when nodal tissues were prepared from seedlings germinated in darkness. These responses were optimal on medium containing 5 μm BA during seedling growth and subsequent culture of explants. The number of shoots per explant was two to five times higher on explants cultured on medium with 0.25 to 1.0 μm forchlorfenuron (CPPU) or thidiazuron (TDZ) than on medium with 5 μm BA. Higher (2.5 and 5 μm) CPPU and TDZ concentrations inhibited shoot elongation and stimulated callus production. Histological analyses indicated that adventitious meristems formed 6 to 8 days after explant culture. Progenies from regenerated plants appeared similar to plants raised from the original seed stocks. Chemical names used: N- (phenylmethyl) -1 H- purin-6-amine (benzyladenine, BA); N- (2-chloro-4-pyridyl)-N'- phenylurea (forchlorfenuron, CPPU); N- phenyl -N' -1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ).
H.M. Ariyarathne, Dermot P. Coyne, and Geunhwa Jung
Halo blight (HB), brown spot (BS), and rust incited by the bacterial pathogens Pseudomonas syringae pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss) and the fungal pathogen Uromyces appendiculatus, respectively, are important diseases of common beans. The objectives were to construct a RAPD linkage map, and to locate HB and BS resistance genes and genes for some other traits. One-hundred-seventy RAPD markers were mapped in 78 RI lines of the cross BelNeb 1 and A 55. Eleven main and nine minor linkage groups were identified. MAPMAKER/QTL, interval mapping, was used to identify genomic regions involved in the genetic control of the traits. One region was found to control HB leaf reactions to strain HB16 while three regions controlled reactions to strain HB 83. These regions accounted for 22% and 18%, 17%, and 17% of phenotypic variation of resistance, respectively. Four putative QTLs were identified for resistance to BS, and accounted for 37%, 26%, 23%, and 19% of the phenotypic variation. Rust resistance was determined by a single major gene to both rust strains US85NP 5-1 and D82vc74fh. However, linked markers were not identified. The V gene controlling flower and stem color was tightly linked with the Operon marker O10.620.